Pathological angiogenesis is a key process that drives vascular remodeling in numerous cardiovascular and metabolic diseases including diabetic retinopathy and cancer. However, due to the lack of appropriate targets, limited effective medicines or therapies to treat this pathological process are available. Epsins were originally identified by the PI as a family of important endocytic adaptor proteins. We recently showed that epsins, via their ubiquitin-interacting motifs (UIM), are critical for activated VEGFR2 internalization, degradation and signal attenuation. Intriguingly, endocytosis of activated VEGFR2 by a different endocytic adaptor protein, Dab2, results in enhanced VEGF signaling. How VEGFR2 internalization differentially modulates VEGF signaling is a highly significant and open question. Our latest study shows that epsins and Dab2 competitively interact with VEGFR2. Furthermore, a synthetic UIM peptide inhibitor interferes with epsin-VEGFR2 association, heightens VEGF signaling and impedes tumor growth. Conversely, a synthetic peptide derived from Dab2 phospho-tyrosine binding (PTB) domain disrupts Dab2-VEGFR2 binding, reduces VEGF signaling and inhibits retinal angiogenesis. Therefore, we hypothesize that epsins and Dab2 differentially modulate VEGF signaling by competitively binding VEGFR2, and that targeting epsins and Dab2 may offer therapeutic potential in pathological angiogenesis. The roles of epsins and Dab2 in pathological angiogenesis and the molecular mechanisms underlying their differential regulation of VEGF signaling will be characterized using both in vitr and in vivo model systems. In Aim 1, we will utilize multifactorial approaches to investigate molecular mechanisms by which epsin UIM specifically interacts with VEGFR2 to control VEGF signaling and angiogenic responses. In Aim 2, we will employ biochemical characterization, dynamic imaging and immunofluorescence to determine how epsins and Dab2 differentially regulate VEGF signaling. These studies mechanistically link the differential regulations of VEGF signaling and function of epsins and Dab2. In Aim 3, we will determine whether targeting epsins and Dab2 disrupts tumor angiogenesis and inhibits neovascular proliferative retinopathy. These studies will advance understanding of new pathways and new targets that differentially modulate VEGF signaling and may lead to new therapies targeting the pathological angiogenesis associated with cancer and diabetes.